Uricase Activity Research Articles

The moose, purine degradation, and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.

A simple and sensitive fluorescence based biosensor for the determination of uric acid using H2O2-sensitive quantum dots/dual enzymes.

A novel optical detection system consisting of combination of uricase/HRP-CdS quantum dots (QDs) for the determination of uric acid in urine sample is described. The QDs was used as an indicator to reveal fluorescence property of the system resulting from enzymatic reaction of uricase and HRP (horseradish peroxidase), which is involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The hydrogen peroxide produced was able to quench the QDs fluorescence, which was proportional to uric acid concentration. The system demonstrated sufficient activity of uricase and HRP at a ratio of 5U:5U and pH 7.0. The linearity of the system toward uric acid was in the concentration range of 125-1000 µM with detection limit of 125 µM.

Uricase-immobilization on radiation grafted polymer support for detection of uric acid using Ag-nanoparticle based optical biosensor

A functional polymer support was designed for covalent immobilization of enzyme uricase, which was further utilized in a single enzyme based silver nanoparticles (Ag-NPs)-optical biosensor system for estimation of uric acid. A carboxylic acid functionalized polymer support was fabricated via gamma radiation induced mutual-irradiation grafting of polyacrylic acid (PAA) on to non-woven polypropylene (PP) matrix. Grafting parameters, such as dose, monomer concentration, homopolymer inhibitor and inorganic acid concentration were studied to optimize the grafting process. PAA-g-PP samples were characterized by grafting yield measurement, water uptake, FTIR, TGA and SEM techniques. Uricase enzyme was immobilized on to the PAA-g-PP support via covalent amide linkage using coupling agents, namely N-hydroxysuccinimide (NHS) and N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide (EDC). The catalytic activity of uricase was investigated spectrophotometrically using uric acid as a substrate. Study of kinetic parameters (Km, Vmax) revealed the value of Km for covalently immobilized uricase to be ∼1.2-fold higher than that of free enzyme. The uricase-immobilized-PAA-g-PP samples could be repeatedly used for ∼20 cycles over a period of 30 days when stored in phosphate buffer (pH∼7.4) at 4°C without significant decrease in the enzyme activity. The uricase immobilized samples were successfully used in conjunction with the radiolytically synthesized Ag-NPs as a LSPR-optical biosensor for estimation of uric acid.

Covalent Immobilization of Uricase Inside a Plastic Vial for Uric Acid Determination in Serum and Urine

Uricase from Candida species was immobilized covalently onto the inner wall of a plasticized polyvinyl chloride (PVC/plastic) vial through glutraldehyde coupling with a 65.23% retention of its initial activity and a conjugation yield of 0.37 mg/cm(2). The vial-bound enzyme showed the optimum activity at pH 7.2, when incubated at 45°C for 5 min. There was a linear relationship between the immobilized uricase activity and the uric acid concentration in the range of 0.01 to 1.2 mM with an apparent Km for uric acid of 0.17 mM. The vial-bound enzyme was employed for an enzymic colorimetric determination of uric acid in serum and urine. The minimum detection limit of the method was 0.01 mM. The analytical recoveries of added uric acid in serum (10 and 20 mM) were 98.0 and 96.5%, respectively. Within and between assays, the coefficients of variation (CVs) for urate in sera determinations were 5.6 and 4.7%, respectively. A good correlation (r = 0.997) was obtained between the serum uric acid values by the standard enzymic colorimetric method using free enzyme and the present method. The vial was reused 200-times over a period of 4 months, when stored at 4°C.

Evolutionary history and metabolic insights of ancient mammalian uricases

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

Activity and stability of uricase from Lactobacillus plantarum immobilizated on natural zeolite for uric acid biosensor.

Determination of uric acid concentration in human urine and blood is needed to diagnose several diseases, especially the occurrence of kidney disease in gout patients. Therefore, it is needed to develop a simple and inexpensive method for uric acid detection. The purpose of the research was to observe the use of Indonesian microbe that was immobilized on natural zeolite as a source of uricase for uric acid biosensor. Selection of mediators and determination of optimum condition measurement, the stability and kinetic properties of L. plantarum uricase were performed using carbon paste electrode. Cyclic voltammetry was employed to investigate the catalytic behavior of the biosensor. The result indicated that the best mediator for measurement of L. plantarum uricase activity was Qo (2,3-dimethoxy-5-methyl-1,4 benzoquinone). Optimum conditions for immobilization of L. plantarum uricase on zeolite were obtained at pH 7.6, with temperature of 28 degrees C, using uric acid concentration of 0.015 mM and zeolite mass at 135 mg K(M) and V(Max) of L. plantarum uricase obtained from Lineweaver-burk equation for the immobilization uricase on zeolite were 8.6728 x 10(-4) mM and 6.3052 mM, respectively. K(M) value of L. plantarum uricase directly immobilized onto the electrode surface was smaller than K(M) value of L. plantarum uricase immobilized on zeolite. The smaller K(M) value shows the higher affinity toward the substrate. The Electrode when kept at 10 degrees C was stable until 6 days, however the immobilized electrode on zeolite was stable until 18 days. Therefore, Indonesian L. plantarum could be used as a uric acid biosensor.

Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients

IntroductionPegloticase, a PEGylated recombinant porcine uricase, is approved for treating refractory gout at a dose of 8 mg intravenous (IV) every 2 weeks. However, during phase 1 testing, pharmacokinetics supported less frequent dosing. Also, single doses of pegloticase unexpectedly induced antibodies (Ab) that bound to polyethylene glycol (PEG). We have conducted a phase 2 trial to evaluate every 3-week dosing, and to further define the Ab response to pegloticase. Organ transplant recipients were included, as they are prone to severe gout that is difficult to manage, and because treatment to prevent graft rejection might influence the immune response to pegloticase.MethodsPlasma uricase activity (pUox), urate concentration (pUA), and clinical response were monitored during up to 5 infusions in 30 patients, including 7 organ transplant recipients. Depending on whether pUA <6 mg/dL was achieved and maintained, patients were classified as non (NR), persistent (PR), or transient (TR) responders. Ab to pegloticase and 10 kDa mPEG were monitored by enzyme linked immunosorbent assay and specificity was further defined.ResultsWe observed 17 PR, 12 TR, and 1 NR; 21 patients (16 PR, 5 TR) received all 5 infusions. Over the 15-week trial, pUA in PR averaged 1.0 ± 0.4 mg/dL; T½ for pUox was approximately 13 days, and area under the curve after dose 5 was approximately 30% higher than after dose 1. PR showed clinical benefit and in some, tophi resolved. In 11 of 12 TR, pUox fell rapidly and hyperuricemia recurred before dose 2. In all TR and NR, loss of response to pegloticase was accompanied by Ab to PEG, which was pre-existing in half of those who had no prior exposure to pegloticase. No PR, and 1 one out of 7 organ transplant recipients, had a sustained Ab response to pegloticase.ConclusionsEvery 3-week dosing is effective and may enhance the utility of pegloticase for treating refractory gout. Ab to PEG, which were pre-existing or induced by treatment, caused rapid loss of efficacy and increased the risk of infusion reactions. Organ transplant recipients can benefit from pegloticase, and may be less prone than non-recipients to developing anti-PEG Ab. Investigation of immunosuppressive strategies to minimize anti-PEG Ab is warranted.Trial registrationClincalTrials.gov identifier: NCT00111657

Xylenol-orange-assay-of-hydrogen-peroxide for Measuring Uricase Activity and Recognizing High-activity Uricase Mutant

This study investigated xylenol-orange-assay-of-hydrogen-peroxide(XOAHP) for measuring uricase activities in cell lysates and recognizing mutants of higher activity.Four mutants of the intracellular uricase of Bacillus fastidiosus were combined into pairs so that ratios of their catalytic capacities varied from 1.3 to 4.1.The pET28a vector containing mutant gene was transformed into Escherichia coli BL21(DE3).Each of thirty clones was transferred into 1.0 mL liquid medium for amplification,and subject to induced expression for 16 h at 18 ℃.Cells were then harvested and lyzed via sonication treatment to get the supernatant as lysate.In Tris-HCl buffer at pH 8.9,XOAHP was effective to measure uricase activity with uric acid of no more than 0.33 mmol/L.SDS-PAGE showed small differences in expression efficiency of mutants.The activity concentration was positively related to the level of total proteins in lysates of tested mutants.Receiver-operation-curve(ROC) analysis showed the higher the ratio of catalytic capacities of uricase mutants in pairs the chloser to 1.00 of the area-under-thecurve(AUC),with an AUC 0.95 for a pair of mutants at the ratio of about 1.8 between their catalytic capacities.With a threshold of the difference between the activity concentration of a candidate as 1.4 times that of the standard deviation of the starting material,positive mutants of catalytic capacities 1.8 times that of the starting material could be effectively recognized.Therefore,XOAHP plus a proper threshold is effective for high-throughput-screening of mutant library of uricase.

FRI0372 Pharmacokinetics and immunogenicity of pegloticase (PL) infused every 3 weeks to treat refractory gout (RG), including in organ transplant recipients (TR)

BackgroundPL is approved for treating RG at 8 mg IV every 2 wks. Phase 1 testing suggested that 8 mg IV every 3 wks might also be effective. We have...

FRI0390 DUKE-3: A 15-week open-label trial of pegloticase in refractory gout including patients with solid organ transplant or prior pegloticase treatment

BackgroundA subset of patients develops refractory gout (RG) characterized by ongoing disease manifestations and inability to control serum urate (SUA) with conventional therapy. Pegloticase (PL) reduces SUA and improves signs...

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model

BackgroundFor screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.ResultsDuring uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity.ConclusionSpecific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Uric acid transporter ABCG2 is increased in the intestine of the 5/6 nephrectomy rat model of chronic kidney disease

Uric acid (UA) remains a risk factor of chronic kidney disease (CKD). Therefore, it is important to clarify the mechanism of UA excretion in CKD. The specific mechanisms of extrarenal excretion from the intestine are unknown. We evaluated the expression of the UA transporter in the intestinal tract--the ATP-binding cassette transporter G2 (ABCG2)--in a 5/6 nephrectomy rat model of CKD. Male Wistar rats (6 weeks old) were randomly assigned to the 5/6 nephrectomized (Nx) group or the sham-operated control group. Urine and blood samples were collected every 4 weeks. All the rats were killed at 8 weeks to obtain liver, duodenum, jejunum, ileum, and transverse colon tissues. Uricase activity was measured in the liver. Expression of ABCG2 in intestinal mucosa was measured with real time polymerase chain reaction (PCR). The Nx group showed significantly decreased urine UA excretion/body weight and UA clearance compared to the control group at 4 and 8 weeks after nephrectomy. In contrast, serum UA and uricase activity were not significant. The expression of ABCG2 in the ileum of the Nx group showed significantly increased upregulation, while no changes were seen in the intestines of the control group. The Nx rats exhibited lower excretion of urine UA and over-expression of ABCG2 in the ileum. The fact that serum UA did not increase despite the decrease in UA excretion suggests that an excretory pathway other than the kidney, probably the intestine, may operate in a complementary role that corroborates the increase in ABCG2 expression in the ileum.

The potential to reduce poultry nitrogen emissions with specific uricase egg yolk feed grade antibodies

One of the environmental challenges that the poultry industry has faced is manure utilisation and/or disposal. Poultry manure and its nitrogenous compounds can be a potential pollutant causing eutrophication, nitrate or nitrite contamination of water, ammonia volatilisation, and acid deposition in the air. Therefore, reducing nitrogen excretion and emissions in poultry manure is important to maintain a clean environment. Proper nutrition is an important first step to optimise performance and growth in animals as well as to reduce the negative impacts on the environment. Amino acids are components of protein nutrition that greatly influence animal growth. However, excess or insufficient amino acid supplementation in diets increases nitrogen excretion and emissions. One way to reduce this is to prevent uricase activity in the microflora in poultry manure. Egg yolk antibodies are economic alternatives for administering in feed to poultry. Supplementation of feed grade antibodies into poultry diets can be a potential approach to minimise bacterial uricase activity and reduce ammonia emissions from poultry manure. In order to use this dietary strategy effectively, a well-balanced feed formulation and a more practical way of delivering the antibodies in feeds remains to be developed.

Isolation, selection and active determination of bacteria produce uricase

Uricase producing bacteria was isolated from soil with a nutrient agar media containing uric acid. The isolation of bacteria was based on uricolytic activity on agar surface. A bacterial strain CN3 showed a maximum zone of clearance in short time had been chosen. Based on morphological and biochemical tests, as well as 16S rRNA sequence and phylogenetic tree analysis, the bacterial isolated was identified as Pseudomonas putida. The uricase was present as intracellular and extracellular enzyme in this strain. When the strain was cultured at 30°C and pH7 for 48h on nutrient broth media containing uric acid 0.5%, the extracellular uricase activity peaked at ~ 0.85 U/ml and intracellular uricase activity peaked at ~ 0.45 U/ml.

Production and characterization of uricase from Streptomyces exfoliatus UR10 isolated from farm wastes

Uricase plays an important role in nitrogen metabolism and can be used medically as a diagnostic reagent. From soil, wastewater, and poultry waste samples collected in Jeddah, 49 bacterial isolates were obtained on either nutrient agar or starch nitrate agar. All the obtained isolates were screened on minimal medium containing 0.5% uric acid for uricase production. The most active bacterium (isolate UR10) produced about 0.5 U/mL of intracellular uricase and was identified as a species belonging to the genus Streptomyces using morphological, physiological, and biochemical characters. By 16S rDNA, it was identified as Streptomyces exfoliatus UR10. Maximum uricase production was obtained using medium 2 with 0.2% uric acid as an inducer, an initial pH of 6.5, and an incubation temperature of 37 °C at 100 rpm. At the end of the incubation period, the cells were collected and disturbed, and the uricase enzyme was precipitated by ammonium sulfate. The enzyme was purified using different column chromatography methods, and the molecular weight of the purified uricase was determined by SDS-PAGE electrophoresis. The optimum temperature for maximum uricase activity was 45 °C; the optimum pH was 8. Co^{2+}, Ni^{2+}, Zn^{2+}, Cu^{2+}, and Pb^{2+} decreased the enzyme activity, whereas Ca^{2+}, Mn^{2+}, Mg^{2+}, and Fe^{2+} stimulated it. In conclusion, uricase was produced by Streptomyces in a medium containing uric acid as inducer, and this enzyme can be used to detect and quantify uric acid in urine and/or blood.

Structure-based characterization of canine–human chimeric uricases and its evolutionary implications

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the β-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure-activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.

Supplementation of Poultry Feeds with Dietary Zinc and Other Minerals and Compounds to Mitigate Nitrogen Emissions—A Review

One of the environmental challenges that the poultry industry has been faced with is ammonia emission from manure. One way to reduce nitrogen excretion and emissions is supplementing dietary trace minerals to inhibit the activity of microbial uricase, a key enzyme converting nitrogen compounds in the manure into ammonia. Several dietary minerals are commercially available as economic alternatives for reducing ammonia emissions in poultry. In this review, we discuss different mineral elements including zinc as feed amendment minerals that could be used to reduce ammonia emission. Issues discussed include potential for inhibiting microbial uricase, dietary supplementation levels, growth performance, toxicity, their influence on manure nitrogen emission, and potential mineral accumulation in soil. In addition, we discuss other minerals and compounds that have the potential to reduce ammonia volatilization by inhibiting microbial uricase and growth of uric acid-utilizing microorganisms.

Hibiscus sabdariffa L. extracts reduce serum uric acid levels in oxonate-induced rats

Elevated serum levels of uric acid are associated with an increased risk for hyperuricemia, gout, hypertension, cardiovascular disease, and renal failure. Recent attention has focused on the bioactive properties of edible plants in preventing disease. Hibiscus sabdariffa L., a local soft drink material and medicinal herb in Taiwan, is used effectively in native medicines against hypertension, pyrexia, and liver disorders. We investigated the effects of the Hibiscus sabdariffa extract (HSE) on oxonic acid (OA)-induced hyperuricemia in rats. The HSE affected serum uric acid levels and urate enzymes such as uricase and xanthine oxidase (XO). We treated rats intraperitoneally with normal saline and oxonate solution for 1week and with or without feeding allopurinol (an XO inhibitor) or HSE (1%, 2%, and 5%) for 5weeks. We observed that treatment with HSE inhibited OA-induced hyperuricemia, with a greater uric acid lowering effect than allopurinol treatment. Our results showed that HSE effectively inhibited OA-induced hyperuricemia by decreasing uric acid and increasing uricase activity, but not by affecting XO activity.

Production and Partial Characterization of Uric Acid Degrading Enzyme from New Source Saccharopolyspora sp. PNR11

The strain PNR11 was isolated from gut of termite during the screening for uric acid degrading actinomyces. This strain was able to produce an intracellular uricase when cultured in fermentation medium containing uric acid as nitrogen source. Base on its morphological characters and 16S rDNA sequence analysis, this strain belong to the genus Saccharopolyspora. This is the first report ofuricase produced from the genus Saccharopolyspora. The aim of this study was to investigate the effects of different factors on uricase production by new source of Saccharopolyspora. Saccharopolyspora sp. PNR11 was cultured in production medium in order to determine the best cultivation period. The result showed that the time period required for maximum enzyme production was 24 h on a rotary shaker operating at 180 rpm. Optimized composition of the production medium consisted of 1% yeast extract, 1% maltose, 0.1% K2HPO4, 0.05% MgSO4 7H2O, 0.05% NaCl and 1% uric acid. The optimum pH and temperature for uricase production in the optimized medium were pH 7.0 and 30 degrees C, respectively. When the strain was cultured at optimized condition, the uricase activity reached to 216 mU mL(-1) in confidential level of 95%. The crude enzyme had an optimum temperature of uricase was 37 degrees C and it was stable up to 30 degrees C at pH 8.5. The optimum pH ofuricase was 8.5 and was stable in range of pH 7.0-10.0 at 4 degrees C. This strain might be considered as a candidate source for uricase production in the further studies. Present finding could be fulfill the information ofuricase produce from actinomycetes.

A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.